DESCRIPTION: The activation of dioxygen in biological systems is a question of great importance because of its involvement in many important metabolic processes. Although the mechanism of oxygen activation in heme systems such as cytochrome P450 and peroxidases is becoming quite well understood, much less is known of the corresponding mechanisms for mononuclear non-heme iron systems. These latter enzymes are involved in the biosynthesis of amino acids, neurochemicals, and antibiotics and carry out reactions that range from oxidative cleavage of catechols, aliphatic and aromatic hydroxylation, to ring cyclizations involving heteroatoms and cis-dihydroxylation of arenes. For many mononuclear nonheme iron enzymes, the reaction with 02 usually occurs only after substrate or cofactor interacts with the active site iron, e.g. catechol for catechol dioxygenases, substrate thiol for isopenicillin N synthase, and the alpha-keto acid co-substrate for alphaketoglutarate- dependent oxygenases. Several of these enzymes have now been shown by crystallography to have an iron center with a common 2- His-1-carboxylate facial triad binding motif. Based on this motif, we have proposed a general mechanism that ties together the chemistry of these systems, but the details of the dioxygen activation step vary from enzyme to enzyme because of the differing natures of the substrates and/or cofactors that interact with the metal center. Important questions include, 1) how is 02 activated by these enzymes, and 2) what is the nature of intermediates that are involved in the reactions? Spectroscopic (NMR, EFR, resonance Raman, Mossbauer, EXAFS, electrospray mass spectrometry) and mechanistic studies are proposed for a number of enzymes to elucidate details of the active site structure and their mechanisms of action. These include extradiol cleaving catechol dioxygenases (both Fe- and Mn-dependent forms), the alpha-ketoglutarate dependent enzymes that oxidize the herbicide 2,4-D and taurine, isopenicillin N synthase, and ACCO, the ethylene forming enzyme in plants. Model complexes will be synthesized to interpret spectroscopic features of the enzymes and their intermediates and mimic the key mechanistic steps of the oxidations. Modeling efforts will focus on: 1) extradiol cleavage of catechols, 2) the role the alpha-keto acid cosubstrate plays in oxygenase reactions, 3) trapping of transient iron-peroxo and high valent iron-oxo intermediates, and 4) correlating the properties of these intermediates with their ability to oxidize hydrocarbon substrates. As in the past, the synergistic interaction of the biochemical and inorganic aspects of the proposal is important for the success of the program.